Analysis of genes induced in human periodontal ligament cells respond to the mechanical stress

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Human PDL tissue was excised from a premolar or a third molar, which were extracted from orthodontic reasons, and placed onto flexible bottom plate with Dalbecco's modified eagle medium (DMEM) containing 10 % fetal bovine serum. After 10 to 14 days fibroblastic cells were extended and after 1 or 3 months later, tension force was applied on the primary culture cells for 24h. The cells derived from the PDL tissue from the same patient without stress were used for control. Total RNA was extracted from both cells and reverse transcribed into cDNA, followed by PCR. The difference of the expression level of 1176 kinds of genes was analyzed by Micro Array method. Same experiments were done with other 3 patient's periodontal tissue and mRNA was collected at some points from early stage to late stage after mechanical stress. The genes reinforced by the mechanical stress, and those suppressed were identified. Some genes expressed strongly in early stage transiently, that decreased with time, and some showed reverse pattern. On the other hand, the mechanical tension force was applied using the cells that were subcultured to 3 passages. The results were almost the same to the experiments using the primary culture cells, although the number of genes responded to the stress were less and the amount of the expression change by stress was smaller. These results suggested that the aging accompanied with the long culture days or subculture lowed down the sensitivity for the mechanical stress or caused the selection for the cells that cannnot respond to the stress.Now, we are preparing the paper about these observations to submit to the international journals, and some of them have been accepted.